Abstract
BackgroundSystemic sclerosis (SSc) is a chronic inflammatory autoimmune disease characterised by vascular dysfunction and damage, excess collagen deposition and subsequent organ manifestations. Vasculopathy is an early feature of the disease which leads to a chronic hypoxic environment in the tissues. Paradoxically, there is a lack of angiogenesis. We hypothesised that this may in part be due to a nonphysiological, overriding upregulation in antiangiogenic factors produced by the hypoxic tissues. We considered thrombospondin 1 (TSP-1) as a candidate antiangiogenic factor.ResultsConditioned media from human microvascular endothelial cells cultured in both normoxic and hypoxic environments were able to block endothelial cell proliferation, with the latter environment having a more profound effect. Filtration to remove > 100-kDa proteins or heparin-binding proteins from the conditioned media eliminated their antiproliferative effect. TSP-1 was expressed in high concentrations in the hypoxic media, as was vascular endothelial growth factor (VEGF). Depletion of TSP-1 from the media by immunoprecipitation reduced the antiproliferative effect. We then show that, in a dose-dependent fashion, recombinant TSP-1 blocks the proliferation of endothelial cells. Immunohistochemistry of skin biopsy material revealed that TSP-1 expression was significantly higher throughout the skin of patients with SSc compared with healthy controls.ConclusionsDespite the environment of chronic tissue hypoxia in SSc, there is a paradoxical absence of angiogenesis. This is thought to be due in part to aberrant expression of antiangiogenic factors, including TSP-1. We have demonstrated that TSP-1 is released in high concentrations by hypoxic endothelial cells. The conditioned media from these cells is able to block proliferation and induce apoptosis in microvascular endothelial cells, an effect that is reduced when TSP-1 is immunoprecipitated out. Further, we have shown that recombinant TSP-1 is able to block proliferation and induce apoptosis at concentrations consistent with those found in the plasma of patients with SSc and that its effect occurs in the presence of elevated VEGF levels. Taken together, these data are consistent with a model wherein injured microvascular cells in SSc fail to repair because of dysregulated induction of TSP-1 in the hypoxic tissues.
Highlights
Systemic sclerosis (SSc) is a chronic inflammatory autoimmune disease characterised by vascular dysfunction and damage, excess collagen deposition and subsequent organ manifestations
Culture under serum-free conditions was used as a negative control, and medium plus 10% foetal calf serum (FCS) was used as a positive control
Cells cultured in media transferred from hypoxic human microvascular endothelial cell 1 (HMEC-1) cells took on an altered morphology with more rounding up of cells (Figure 1)
Summary
Systemic sclerosis (SSc) is a chronic inflammatory autoimmune disease characterised by vascular dysfunction and damage, excess collagen deposition and subsequent organ manifestations. We hypothesised that this may in part be due to a nonphysiological, overriding upregulation in antiangiogenic factors produced by the hypoxic tissues. Vasculopathy occurs early in the disease and precedes fibrosis [2]. This is characterised by both abnormal vascular tone and endothelial cell damage [3]. Anti-endothelial cell antibodies have been detected in some but not all patients with SSc [5]. These antibodies are capable of upregulating the expression of endothelial cell adhesion molecules and inducing apoptosis [6]
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